Fortimicin factors KG1, KG2 and KG3 and processes for production thereof

ABSTRACT

New antibiotic compounds, Fortimicin factors KG 1 , KG 2  and KG 3  are produced by fermentation of microorganisms belonging to the genus Micromonospora. The antibiotic compounds are accumulated in the culture liquor and are isolated therefrom.

RELATED APPLICATIONS

The present invention is related generally to the inventions disclosed in U.S. Pat. No. 3,931,400 issued Jan. 6, 1976 for Fortimicin B and Process For Production Thereof; U.S. Pat. No. 3,976,768 issued Aug. 24, 1976 for Fortimicin A and Process For Production Thereof; U.S. Pat. No. 4,048,015 issued Sept. 13, 1977 for Fortimicin C and Process for Production Thereof; U.S. Pat. application Ser. No. 845,970, filed Oct. 27, 1977, now U.S. Pat. No. 4,145,253, for Fortimicin Factors D and KE and Processes for Production Thereof; and U.S. patent application Ser. No. 957,845, filed Nov. 6, 1978 for Fortimicin Factors KF and KG and Processes for Production Thereof.

BACKGROUND OF THE INVENTION

The present invention relates to new compositions of matter having antibacterial properties, namely Fortimicin KG₁, Fortimicin KG₂ and Fortimicin KG₃. The invention also pertains to the production of Fortimicin KG₁, Fortimicin KG₂ and Fortimicin KG₃ by culturing a microorganism belonging to the genus Micromonospora, which is capable of producing at least one of the active substances in a nutrient medium, until antibacterial activity is detected in the culture liquor and then isolating at least one of the active substances therefrom.

Antibiotics which exhibit activity against a broad spectrum of bacteria are always in demand. To this end, it has been found that when certain strains of Micromonospora are cultured in a nutrient medium, several antibiotic substances are produced in the culture liquor. Specifically, Fortimicin factors A, B, C, D, KE, KF and KG have been isolated from the culture liquor of Micromonospora olivoasterospora MK-70 (ATCC 21819) (FERM-P No. 1560) and have the following structural formulae: ##STR1##

The chemical, physical and biological properties of these antibiotics and the processes for the production thereof are explained in detail in the specifications of the aforementioned United States Patents and Patent applications.

It has now been found that Micromonospora olivoasterospora MK-70, when cultured, liberates three further active substances. A study of the chemical, physical and biological properties of these active substances indicates that the compositions of matter are new antibiotics which have now been named Fortimicin KG₁, Fortimicin KG₂ and Fortimicin KG₃.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel antibiotics, Fortimicin factors KG₁, KG₂ and KG₃, are produced by fermentation of a microorganism belonging to the genus Micromonospora which is capable of producing at least one of said factors, in a nutrient medium until substantial antibacterial activity is detected in the culture liquor. At the completion of culturing, the active fractions containing at least one of Fortimicin factors KG₁, KG₂ and KG₃ are isolated from the culture liquor by known means such as by ion exchange resin treatment.

Fortimicin factors KG₁, KG₂ and KG₃ exhibit broad antibacterial activity, and are, therefore, useful inter alia to clean and sterilize laboratory glassware and surgical instruments, and may also be used in combination with soaps, detergents and wash solutions for sanitation purposes. Further, Fortimicin factors KG₁, KG₂ and KG₃ are applicable to medicinal purpose.

Included in the composition of matter aspect of the invention are the pharmaceutically acceptable non-toxic acid addition salts of Fortimicin factors KG₁, KG₂ and KG₃ including the mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, carbonate and nitrate and the organic acid addition salts such as acetate, fumarate, malate, citrate, mandelate, ascorbate, tartarate, succinate and the like.

Such a salt means mono, di, tri or tetra salt formed by the reaction of 1 molecule of Fortimicin factors KG₁, KG₂ or KG₃ with 1-4 equivalent of a pharmaceutically acceptable non-toxic acid.

DETAILED DESCRIPTION OF THE INVENTION

The physicochemical properties of the free base of Fortimicin KG₁ of the present invention are as follows:

(1) A basic white powder

(2) The elementary analytical value found:

C=52.09%, H=8.81%, N=16.23%.

(3) Melting point: 95°-98° C.

(4) Ultraviolet absorption spectrum:

Ultraviolet absorption spectrum of an aqueous solution of the substance does not show characteristic maximum absorption between 220 nm and 360 nm but only shows terminal absorption.

(5) Specific rotation:

[α]_(D) ²² =+58.3° (c=0.66, H₂ O).

(6) Infrared absorption spectrum:

The infrared absorption spectrum is measured in KBr tablet. The free base of Fortimicin KG₁ shows maximum absorption at the following wavenumbers (cm⁻¹): 3360, 2930, 1675, 1590, 1370, 1110, 1055, 1000.

(7) Color reactions:

Ninhydrin reaction: positive

Potassium permanganate reaction: positive

Elson-Morgan's reaction: negative

Biuret reaction: negative

(8) The CMR spectrum of Fortimicin KG₁ is measured in a deuterium oxide solution (pD=10.7) by using JEOL JNM-100A. The results are as follows:

δ (ppm): 153.1, 101.2, 95.2, 83.4, 83.2, 75.2, 73.4, 62.2, 60.1, 54.8, 48.9, 47.4, 33.6, 25.7, 20.5.

(9) The mass spectrum of the substance reveals the following M ion and fragment ions. The formula in parentheses means the composition formula obtained by high resolution mass spectrometry.

m/e: 346.2218 (M⁺) (C₁₅ H₃₀ N₄ O₅), 247.1543 (C₁₀ H₂₁ N₃ O₄), 235.1248 (C₉ H₁₉ N₂ O₅), 207.1318 (C₈ H₁₉ N₂ O₄), 172.0938 (C₈ H₁₄ NO₃).

From the result of the mass spectrometry, the molecular weight of the substance is determined to be 346 and the molecular formula is determined to be C₁₅ H₃₀ N₄ O₅. The elementary analytical values of the substance as calculated from the molecular formula are C=52.00%, H=8.73% and N=16.17%.

(10) Based on the foregoing physicochemical data, the structural formula of Fortimicin KG₁ is considered to be as follows: ##STR2## (11) The free base of Fortimicin KG₁ is readily soluble in water, soluble in methanol and slightly soluble in ethanol and acetone but is insoluble in organic solvents such as chloroform, benzene, ethyl acetate, butyl acetate, ethyl ether, butanol, petroleum ether, n-hexane, etc.

The physicochemical properties of the free base of Fortimicin KG₂ of the present invention are as follows:

(1) A basic white powder

(2) The elementary analytical value found:

C=52.05%, H=8.80%, N=16.21%.

(3) Melting point: 83°-85° C.

(4) Ultraviolet absorption spectrum:

Ultraviolet absorption spectrum of an aqueous solution of the substance does not show characteristic maximum absorption between 220 nm and 360 nm but only shows terminal absorption.

(5) Specific rotation:

[α]_(D) ²² =+30° (C=0.76, H₂ O).

(6) Infrared absorption spectrum:

The infrared absorption spectrum is measured in KBr tablet. The free base of Fortimicin KG₂ shows maximum absorption at the following wavenumbers (cm⁻¹): 3350, 2920, 1675, 1590, 1370, 1090, 1030, 990.

(7) Color reactions:

Ninhydrin reaction: positive

Potassium permanganate reaction: positive

Elson-Morgan's reaction: negative

Biuret reaction: negative

(8) The CMR spectrum of the substance is measured in a deuterium oxide solution (pD=10.6) by using JEOL JNM-100A. The results are as follows:

δ (ppm): 153.1, 101.3, 95.3, 82.2, 80.0, 71.3, 71.1, 61.1, 59.3, 53.8, 48.9, 47.3, 35.4, 25.6, 20.5.

(9) The mass spectrum of the substance reveals the following M ion and fragment ions. The formula in parentheses means the composition formula obtained by high resolution mass spectrometry.

m/e: 346.2244 (M⁺) (C₁₅ H₃₀ N₄ O₅), 247.1528 (C₁₀ H₂₁ N₃ O₄), 235.1301 (C₉ H₁₉ N₂ O₅), 207.1357 (C₈ H₁₉ N₂ O₄), 172.0981 (C₈ H₁₄ NO₃).

From the result of the mass spectrometry, the molecular weight of the substance is determined to be 346 and the molecular formula is determined to be C₁₅ H₃₀ N₄ O₅. The elementary analytical values of the substance as calculared from the molecular formula are C=52.00%, H=8.73% and N=16.17%.

(10) Based on the foregoing physicochemical data, the structural formula of Fortimicin KG₂ is considered to be as follows: ##STR3## (11) The free base of Fortimicin KG₂ is readily soluble in water, soluble in methanol and slightly soluble in ethanol and acetone but is insoluble in organic solvents such as chloroform, benzene, ethyl acetate, butyl acetate, ethyl ether, butanol, petroleum ether, n-hexane, etc.

As is apparent from the foregoing, Fortimicin factors KG₁, KG₂ and KG (U.S. patent application Ser. No. 957,845, filed Nov. 6, 1978) have the same planer formulae. However, these factors differ from one another in melting point, specific rotation, infrared absorption spectrum and CMR spectrum and therefore, these factors are considered to be stereoisomers with one another. For reference, several physicochemical properties of the free base of Fortimicin KG are shown below:

(1) Melting point: 72°-74° C.

(2) Specific rotation:

[α]_(D) ²⁴ =+90° (c=0.33, H₂ O).

(3) Infrared absorption spectrum:

The infrared absorption spectrum is measured in KBr tablet. The free base of Fortimicin KG shows maximum absorption at the following wavenumbers (cm⁻¹): 3350, 1678, 1590, 1448, 1365, 1110.

(4) The CMR spectrum of the substance is measured in a deuterium oxide solution (pD=10.6) by using JEOL PFT-100A. The results are as follows:

δ (ppm): 154.1, 101.2, 94.6, 84.5, 79.7, 73.9, 73.3, 62.8, 62.2, 53.4, 48.8, 47.4, 33.9, 25.8, 21.2.

The physicochemical properties of the free base of Fortimicin KG₃ of the present invention are as follows:

(1) A basic white powder

(2) The elementary analytical value found:

C=50.68%, H=8.29%, N=17.45%.

(3) Melting point: 135°-138° C.

(4) Ultraviolet absorption spectrum:

Ultraviolet absorption spectrum of an aqueous solution of the substance does not show characteristic maximum absorption between 220 nm and 360 nm but only shows terminal absorption.

(5) Specific rotation:

[α]_(D) ²⁰ =+185° (c=0.265, H₂ O).

(6) Infrared absorption spectrum:

The infrared absorption spectrum is measured in KBr tablet. The free base of Fortimicin KG₃ shows maximum absorption at the following wavenumbers (cm⁻¹): 3370, 2940, 1640, 1580, 1462, 1110.

(7) Color reactions:

Ninhydrin reaction: positive

Potassium permanganate reaction: positive

Elson-Morgan's reaction: negative

Biuret reaction: negative

(8) The CMR spectrum of the substance is measured in a deuterium oxide solution containing deuteriochloric acid (pD=1.2) by using JEOL JNM-FX100 The results are as follows:

δ (ppm): 168.8, 146.7, 99.5, 95.9, 76.1, 73.9, 71.4, 66.5, 57.3, 54.2, 52.9, 49.6, 47.2, 41.4, 32.8, 22.2, 16.9.

(9) The mass spectrum of the substance reveals the following M ion and fragment ions. The formula in parentheses means the composition formula obtained by high resolution mass spectrometry.

m/e: 403.2490 (M⁺) (C₁₇ H₃₃ N₅ O₆), 304.1750 (C₁₂ H₂₄ N₄ O₅), 264.1534 (C₁₀ H₂₂ N₃ O₅), 246.1430 (C₁₀ H₂₀ N₃ O₄), 100.0723 (C₅ H₁₀ NO).

From the result of the mass spectrometry, the molecular weight of the substance is determined to be 403 and the molecular formula is determined to be C₁₇ H₃₃ N₅ O₆. The elementary analytical values of the substance as calculated from the molecular formula are C=50.61%, H=8.24%, N=17.36%.

(10) Based on the foregoing physicochemical data, the structural formula of Fortimicin KG₃ is considered to be as follows: ##STR4## (11) The free base of Fortimicin KG₃ is readily soluble in water, soluble in methanol and slightly soluble in ethanol and acetone but is insoluble in organic solvents such as chloroform, benzene, ethyl acetate, butyl acetate, ethyl ether, butanol, petroleum ether, n-hexane, etc.

As apparent from the foregoing, one aspect of the present invention relates eventually to a composition of matter having antibacterial activity which is represented by the general formula I: ##STR5## wherein R is ##STR6## or H and when R is H said composition of matter has the following physicochemical properties:

(1) Melting point: 95°-98° C.,

(2) Specific rotation: [α]_(D) ²² =+58.3° (c=0.66, H₂ O),

(3) Infrared absorption spectrum (KBr): 3360, 2930, 1675, 1590, 1370, 1110, 1055 and 1000 cm⁻¹ and

(4) The CMR spectrum [deuterium oxide solution (pD=10.7)]:

δ (ppm): 153.1, 101.2, 95.2, 83.4, 83.2, 75.2, 73.4, 62.2, 60.1, 54.8, 48.9, 47.4, 33.6, 25.7, 20.5.

or the following physicochemical properties:

(1) Melting point: 83°-85° C.,

(2) Specific rotation: [α]_(D) ²² =+30° (c=0.76, H₂ O),

(3) Infrared absorption spectrum (KBr): 3350, 2920, 1675, 1590, 1370, 1090, 1030 and 990 cm⁻¹ and

(4) The CMR spectrum [deuterium oxide solution (pD=10.6)]:

δ (ppm): 153.1, 101.3, 95.3, 82.2, 80.0, 71.3, 71.1, 61.1, 59.3, 53.8, 48.9, 47.3, 35.4, 25.6, 20.5.

and the pharmaceutically acceptable non-toxic acid addition salts thereof.

The Rf values of Fortimicin factors KG₁, KG₂ and KG₃ in paper chromatography and thin layer chromatography are shown in the following Tables 1 and 2. For comparison, the Rf values of antibiotics similar to Fortimicin factors KG₁, KG₂ and KG₃ are also given.

                  Table 1                                                          ______________________________________                                         The Rf values in ascending paper chromatography                                using as developer the lower layer of chloroform,                              methanol and concentrated aqueous ammonia (2:1:1                               by volume) (at room temperature; after four hours                              of development)                                                                Antibiotic      Rf value                                                       ______________________________________                                         Fortimicin A    0.41                                                           Fortimicin B    0.72                                                           Fortimicin C    0.22                                                           Fortimicin D    0.22                                                           Fortimicin KE   0.63                                                           Fortimicin KF   0.49                                                           Fortimicin KG   0.67                                                           Fortimicin KO*  0.53                                                           Fortimicin KG.sub.1                                                                            0.66                                                           Fortimicin KG.sub.2                                                                            0.70                                                           Fortimicin KG.sub.3                                                                            0.28                                                           ______________________________________                                          *Fortimicin KO is disclosed in Japanese Patent application No.                 113193/1977, filed September 21, 1977 and has the following structural         formula.                                                                       ##STR7##                                                                 

                  Table 2                                                          ______________________________________                                         The Rf values in silica gel thin layer chromatography                          (at room temperature; after three hours of develop-                            ment; Kieselgel 60 by E. Merck & Co. Inc. is used)                                        Rf value                                                            Antibiotic   Developer I* Developer II**                                       ______________________________________                                         Fortimicin A 0.36         0.09                                                 Fortimicin B 0.75         0.13                                                 Fortimicin C 0.23         0.19                                                 Fortimicin D 0.21         0.06                                                 Fortimicin KE                                                                               0.50         0.12                                                 Fortimicin KF                                                                               0.35         0.12                                                 Fortimicin KG                                                                               0.55         0.12                                                 Fortimicin KO                                                                               0.55         0.24                                                 Fortimicin KG.sub.1                                                                         0.58         0.14                                                 Fortimicin KG.sub.2                                                                         0.70         0.20                                                 Fortimicin KG.sub.3                                                                         0.32         0.11                                                 ______________________________________                                          *Developer I: The lower layer of chloroform, methanol and concentrated         aqueous ammonia (2:1:1 by volume)                                              **Developer II: 10% (W/V) aqueous ammonium acetate, methanol and               concentrated aqueous ammonia (50:50:1 by volume)                         

Table 3 illustrates the antibacterial spectra of Fortimicin factors KG₁, KG₂ and KG₃ against various microorganisms.

                  Table 3                                                          ______________________________________                                         (Minimum Inhibitory Concentration, μg/ml measured by                        agar dilution method at pH 8.0)                                                                  Forti-   Forti-   Forti-                                                       micin    micin    micin                                      Microorganism     KG.sub.1 KG.sub.2 KG.sub.3                                   ______________________________________                                         Bacillus subtilis No. 10707                                                                      --       --       <0.045                                     Staphylococcus aureus                                                                            13.1     10.5      0.083                                     ATCC 6538P                                                                     Klebsiella pneumoniae                                                                            --       --       0.18                                       ATCC 10031                                                                     Escherichia coli ATCC 26                                                                         26.1     41.7     0.33                                       Escherichia coli KY8302                                                                          26.1     41.7     0.52                                       (resistant to chloramphenicol,                                                 streptomycin, kanamycin,                                                       paromomycin, tetracycline                                                      and spectinomycin)                                                             Escherichia coli KY8327                                                                          26.1     20.9     0.33                                       (resistant to kanamycin,                                                       gentamicin and tobramycin)                                                     Escherichia coli KY8348                                                                          26.1     166.6    10.4                                       (resistant to streptomycin                                                     and gentamicin)                                                                Proteus vulgaris ATCC 6897                                                                       26.1     83.3     0.66                                       Shigella sonnei ATCC 9290                                                                        --       --       0.7                                        Salmonella typhosa ATCC 9992                                                                     --       --       0.18                                       Pseudomonas aeruginosa BMH#1                                                                     >208     >166.6   5.2                                        ______________________________________                                    

As is apparent from the above, Fortimicin KG₃ has a strong antibacterial activity against a broad range of Gram-positive and Gram-negative bacteria. Particularly, it is characteristic that the antibiotic is effective against certain strains of Escherichia coli which are resistant to various antibiotics. As is also apparent from the above, Fortimicin factors KG₁ and KG₂ exhibit a broad antibacterial spectrum. Therefore, Fortimicin factors KG₁, KG₂ and KG₃, especially Fortimicin KG₃ are expected to have a therapeutic effect on various infections (in human beings and in animals) induced by various bacteria. With such antibacterial properties, Fortimicin factors KG₁, KG₂ and KG₃ are applicable to medicinal purposes. LD₅₀ value of Fortimicin KG₃ (free base) is determined to be 225 mg/kg in mice by intraveneous administration.

Thus the invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, Fortimicin factor KG₁, KG₂ or KG₃, or a pharmaceutically acceptable non-toxic acid addition salt thereof in association with a pharmaceutical carrier or diluent. The compounds of this invention are administered by parenteral (intramuscular, intraperitoneal, intravenous, intramuscular or subcutaneous injection routes) or rectal routes of administration and can be formulated in dosage forms appropriate for each route of administration.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by beating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Compositions for rectal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 5 to 300 mg/kg of body weight daily are administered to mammalian patients to achieve an antibiotic effect.

Fortimicin factors KG₁, KG₂ and KG₃ are produced by fermentation of a microorganism belonging to the genus Micromonospora. Any strain belonging to the genus Micromonospora and capable of forming at least one of Fortimicin factors KG₁, KG₂ and KG₃ in the culture liquor may be used. Examples of preferred strains are Micromonospora olivoasterospora MK-70 (FERM-P No. 1560) (ATCC 21819), Micromonospora olivoasterospora MK-80 (FERM-P No. 2192) (ATCC 31010) and Micromonospora olivoasterospora Mm 744 (FERM-P No. 2193) (ATCC 31009). These strains have been deposited with the American Type Culture Collection, Rockville, Maryland, U.S.A. and with the Fermentation Research Institute Agency of Industrial Science and Technology, Chiba-ken, Japan and have been accorded the accession number noted above.

The microbiological properties of these strains are described in U.S. Pat. No. 3,931,400, which description is expressly incorporated herein by reference.

As in the case with other strains of Actinomycetes, the microorganisms useful in carrying out the present invention can be mutated by artificial means such as ultraviolet irradiation, X-ray irradiation and use of various mutation including chemicals in known manner to enhance the production of metabolic products, an example of which is Micromonospora olivoasterospora CS-26 (FERM-P No. 3567, NRRL 8178). This latter mutant has been deposited with the United States Department of Agriculture, Peoria, Illinois, and is freely available to the public.

Generally, conventional methods for culturing Actinomycetes may be employed in the process of the present invention. Thus, various nutrient sources may be used for the culture medium. Appropriate carbon sources include glucose, starch, mannose, fructose, sucrose, molasses, etc. either alone or in combination. Hydrocarbons, alcohols, organic acids, etc. may also be used depending upon the assimilability possessed by the microorganisms to be used. As inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, may be used either alone or in combination or natural nitrogen sources such as peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid, soluble vegetable protein, etc. are appropriate. If necessary, inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, phosphates, etc. may be added to the medium. Moreover, organic and inorganic materials which promote the growth of the particular strain and the production of at least one of Fortimicin factors KG₁, KG₂ and KG₃ may be added.

A liquid culturing method, particularly a submerged stirring culturing method is most suitable. Culturing temperature is desirably 25°-40° C., and it is preferred to carry out culturing at around neutral pH. Usually, after 2 to 15 days of liquid culturing, Fortimicin factors KG₁, KG₂ and KG₃ are formed and accumulated in the culture liquor. When substantial antibacterial activity is detected in the culture liquor, preferably when the yield of the antibiotics in the culture liquor reaches a maximum, culturing is discontinued and the desired product is isolated and purified from the culture liquor after the microbial cells have been removed such as by filtration.

Isolation and purification of Fortimicin factors KG₁, KG₂ and KG₃ is carried out by methods usually used for the isolation and purification of microbial metabolic products from a culture liquor.

Since the antibiotics fortimicin KG₁, Fortimicin KG₂ and Fortimicin KG₃ are basic substances and are readily soluble in water but poorly soluble in the ordinary organic solvents, the antibiotics can be purified by the methods usually used for the purification of so-called water-soluble basic antibiotics. More specifically, the KG₁, KG₂ and KG₃ factors can be purified by a proper combination of adsorption and desorption from cation exchange resin, cellulose column chromatography, adsorption and desorption using a column of Sephadex LH-20, silica gel column chromatography, etc. As an example, a suitable method of purification of Fortimicin factors KG.sub., KG₂ and KG₃ from the culture liquor when a strain capable of producing the Fortimicin complex (a mixture containing Fortimicin factors A, B, C, D, KE, KF, KG and KO and by-products having antibacterial activity) is used is as follows. The cell-free culture filtrate is adjusted to pH 7.5 and is then flowed through a cation exchange resin such as Amberlite IRC-50 (ammonium form) (Rohm & Haas Co., U.S.A.). After the resin is washed with water, elution is carried out with 0.5 N aqueous ammonia. The active fractions are combined and concentrated under reduced pressure. The concentrate is then flowed through a cation exchange resin, Amberlite CG-50 (ammonium form) (Rohm & Haas Co., U.S.A.). After the resin is washed with water, elution is carried out with 0.1 N aqueous ammonia containing 0.1 M NH₄ Cl. First, Fortimicin factors KO and C are eluted and subsequently Fortimicin factors KG₁, KG₂ and KG₃ are eluted together with Fortimicin B. Thereafter, Fortimicin factors A, D, KE, KF and KG are eluted. The fractions containing Fortimicin KG₁, KG₂ and KG₃ are collected, neutralized with hydrochloric acid and flowed through a cation exchange resin such as Amberlite IRC-50 (ammonium form). After the resin is washed with water, elution is carried out with 0.5 N aqueous ammonia. The active fractions are collected and concentrated to dryness to obtain a crude powder containing Fortimicin factors KG₁, KG₂ and KG₃. The crude powder is placed on the upper part of a column packed with silica gel. Development is carried out with the lower layer of a mixed solvent of chloroform, methanol and concentrated aqueous ammonia (3:1:1 by volume). First, Fortimicin B which is contained in the crude powder is eluted and subsequently Fortimicin factors KG₂, KG₁ and KG₃ are eluted in this order. The fractions containing respectively Fortimicin factors KG₁, KG₂ and KG₃ are collected, concentrated under reduced pressure and freeze-dried to obtain a respective white free base of Fortimicin factors KG₁, KG₂ and KG₃.

Though fairly pure Fortimicin factors KG₁, KG₂ and KG₃ are obtained by the above treatment, these compounds yet contain a small amount of impurities. Therefore, these compounds are respectively flowed through a cation exchange resin, Bio-Rex-70 (ammonium form) [Bio-Rad Laboratories (U.S.A.)]. After the resin is washed with water, elution is carried out with 0.04 N aqueous ammonia containing 0.04 M ammonium acetate. The active fractions are collected, neutralized and flowed through Amberlite CG-50 (ammonium form). After the resin is washed with water, elution is carried out with 0.5 N aqeuous ammonia. The active fractions are collected, concentrated and freeze-dried to obtain a respective pure preparate (free base) of Fortimicin factors KG₁, KG₂ and KG₃.

During the above-described purification procedures, the fractions are checked by silica gel thin layer chromatography. As the developer, the lower layer of a mixed solvent of chloroform, methanol and concentrated aqueous ammonia (2:1:1 by volume) and a mixed solvent of 10% (w/v) aqueous ammonium acetate, methanol and concentrated aqueous ammonia (50:50:1 by volume) are used. Detection is carried out by coloring method using ninhydrin and bioautography using Bacillus subtilis No. 10707 as the test microorganism. Rf values of Fortimicin factors KG₁, KG₂ and KG₃ on the silica gel thin layer chromatogram are shown in Table 2.

Certain specific embodiments of the present invention are illustrated by the following representative examples.

EXAMPLE 1 A. Culturing of the MK-70 strain

Micromonospora olivoasterospora MK-70 (ATCC 21819) (FERM-P No. 1560) is used as the seed strain. A medium comprising 2 g/dl glucose, 0.5 g/dl peptone, 0.5 g/dl yeast extract and 0.1 g/dl calcium carbonate (pH 7.5 before sterilization) is used as a first seed medium. A loopful of the seed strain is inoculated into 10 ml portions of the first seed medium in 50 ml-large test tubes and is cultured at 30° C. for 5 days. Then, 10 ml of the thus prepared first seed culture is added to 30 ml portions of a second seed medium in 250 ml-Erlenmeyer flasks. The second seed medium has the same composition as that of the first seed medium. The second seed culturing is carried out with shaking at 30° C. for 2 days, and thereafter, 30 ml of the second seed culture is added to 300 ml portions of a third seed medium in 2 l-Erlenmeyer flasks provided with baffles. The third seed medium is of the same composition as that of the first seed medium. The third seed culturing is carried out with shaking at 30° C. for 2 days. Then, 1.5 l of the third seed culture (corresponding to 5 flasks) is added to 15 l of a fourth seed medium in a 30 l-stainless steel jar fermenter. The fourth seed medium has the same composition as that of the first seed medium. The fourth seed culturing in the jar fermenter is carried out with aeration and stirring (revolution: 350 r.p.m.; aeration: 15 l/min) at 37° C. for 2 days. Then, 15 l of the fourth seed culture is added to 150 l of a fermentation medium in a 300 l-fermenter. The fermentation medium has the following composition:

    ______________________________________                                         Soluble starch                                                                            4 g/dl  K.sub.2 HPO.sub.4                                                                          0.05 g/dl                                                                             CaCO.sub.3                               Soybean meal                                                                              2 g/dl  MgSO.sub.4 . 7H.sub.2 O                                                                    0.05 g/dl                                                                             0.1 g/dl                                 Corn steep liquor                                                                         1 g/dl  KCl         0.03 g/dl                                       (pH 7.5 before sterilization)                                                  ______________________________________                                    

Fermentation in the fermenter is carried out with aeration and stirring (revolution: 150 r.p.m.; aeration: 80 l/min) at 37° C. for 4 days.

B. Isolation of crude Fortimicin complex containing KG₁, KG₂ and KG₃

After the completion of fermentation, the culture liquor is adjusted to pH 2.5 with concentrated sulfuric acid and is stirred for 30 minutes. Thereafter, about 7 kg of a filter aid, Radiolite No. 600 (product of Showa Kagaku Kogyo Co., Ltd., Japan) is added to the culture liquor and the microbial cells are removed by filtration. The filtrate is adjusted to pH 7.5 by the addition of 6 N sodium hydroxide, and then flowed through a column packed with about 20 l of a cation exchange resin, Amberlite IRC-50 (ammonium form). The active principles are adsorbed on the resin, and the effluent is discarded. After the resin is washed with water, elution of the active principles is carried out with 1 N aqueous ammonia. The eluate is subjected to determination of activity by a paper disc method using an agar plate of Bacillus subtilis No. 10707. Fractions showing an activity are combined and concentrated under reduced pressure to about 1 l. The concentrate is adjusted to pH 7 with concentrated hydrochloric acid and then flowed through a column packed with 3 l of Amberlite CG-50 (ammonium form). After the resin is washed with water, elution is carried out with 0.1 N NH₄ OH containing 0.1 M NH₄ Cl. The eluate is taken in 500 ml fractions. Each fraction is subjected to determination of antibacterial activity against Bacillus subtilis No. 10707 and identification of active component by silica gel thin layer chromatography [Developer: the lower layer of a mixed solvent of chloroform, methanol and concentrated aqueous ammonia (2:1:1 by volume), Coloring agent: ninhydrin]. After the volume of the eluate reaches about 3 l, Fortimicin factors KG₁, KG₂ and KG₃ begin to be eluted. The fractions containing Fortimicin factors KG₁, KG₂ and KG₃ are collected, concentrated under reduced pressure to remove ammonia, neutralized with concentrated hydrochloric acid and flowed through a column packed with 500 ml of Amberlite IRC-50 (ammonium form). After the resin is washed with water, elution is carried out with 0.5 N aqueous ammonia. The active fractions are collected, concentrated under reduced pressure and freeze-dried to obtain 2 g of a white powder. The powder contains in it 250 mg of Fortimicin KG₁, 640 mg of Fortimicin KG₂ and 330 mg of Fortimicin KG₃.

C. Respective isolation and purification of Fortimicin factors KG₁, KG₂ and KG₃

2 g of the crude powder is placed on a silica gel column (Silica gel: produced by Wako Junyaku Co., Ltd., Japan, Diameter of column: about 3 cm). Development is carried out with the lower layer of a mixed solvent of chloroform, methanol and concentrated aqueous ammonia (3:1:1 by volume). The eluate is taken in 20 ml fractions. Each fraction is subjected to the silica gel thin layer chromatography. First, Fortimicin B is eluted and then Fortimicin factors KG₂, KG₁ and KG₃ are eluted in this order. The fractions respectively containing Fortimicin factors KG₁, KG₂ and KG₃ are collected, concentrated under reduced pressure and freeze-dried to obtain 200 mg of Fortimicin KG₁, [Activity: 970 u/mg (u: unit)], 500 mg of Fortimicin KG₂ (Activity: 970 u/mg) and 250 mg of Fortimicin KG₃ (Activity: 950 u/mg) (Activity of each of the purified substances is considered to be 1000 u/mg).

200 mg of the thus obtained Fortimicin KG₁ is dissolved in water. The solution is adjusted to pH 7.0 and flowed through a column packed with 50 ml of Bio-Rex-70 (ammonium form). After the resin is washed with water, elution is carried out with 0.04 N aqueous ammonia containing 0.04 M ammonium acetate. The eluate is taken in 10 ml of fractions and each fraction is subjected to the silica gel thin layer chromatography. The fractions containing only Fortimicin KG₁ are collected, concentrated under reduced pressure to remove ammonia, adjusted to pH 7.0 and flowed through a column packed with 100 ml of Amberlite IRC-50 (ammonium form). After the resin is washed with water, elution is carried out with 0.5 N aqueous ammonia. The active fractions are collected, concentrated under reduced pressure and freeze-dried to obtain 150 mg of a free base of pure Fortimicin KG₁ as a white powder.

500 mg of Fortimicin KG₂ and 250 mg of Fortimicin KG₃ respectively obtained by the above silica gel chromatography are treated in the same manner as above-described with Fortimicin KG₁ to obtain 470 mg of a free base of pure Fortimicin KG₂ and 230 mg of a free base of pure Fortimicin KG₃ respectively as a white powder.

EXAMPLE 2

Micromonospora olivoasterospora Mm 744 (ATCC 31009) (FERM-P No. 2193) is used as the seed strain. A medium comprising 2 g/dl glucose, 0.5 g/dl peptone, 0.3 g/dl yeast extract and 0.1 g/dl calcium carbonate (pH 7.2 before sterilization) is used as a first seed medium. The seed culture (the first seed culture--the fourth seed culture) is carried out in the same manner as described in the Example 1. The composition of the second seed medium--the fourth seed medium is the same as that of the first seed medium. 15 l of the thus obtained fourth seed culture is added to 150 l of a fermentation medium in a 300 l-stainless steel fermenter. The fermentation medium has the following composition:

    ______________________________________                                         Soluble starch                                                                             2 g/dl    K.sub.2 HPO.sub.4                                                                           0.05 g/dl                                   Soybean meal                                                                               0.5 g/dl  MgSO.sub.4 . 7H.sub.2 O                                                                     0.05 g/dl                                   Glucose     2 g/dl    KCl          0.03 g/dl                                   Corn steep liquor                                                                          1 g/dl    CaCO.sub.3   0.1 g/dl                                    Yeast extract                                                                              1 g/dl                                                             (pH 7.0 before sterilization)                                                  ______________________________________                                    

Fermentation in the fermenter is carried out with aeration and stirring (revolution: 150 r.p.m.; aeration: 80 l/min) at 30° C. for 4 days.

After the completion of fermentation, the culture liquor is subjected to the same purification and isolation method as described in the Example 1 to obtain 120 mg of a free base of pure Fortimicin KG₁, 350 mg of a free base of pure Fortimicin KG₂ and 180 mg of a free base of pure Fortimicin KG₃.

EXAMPLE 3

Micromonospora olivoasterospora MK 80 (ATCC 31010) (FERM-P No. 2192) is used as the seed strain. A medium comprising 1 g/dl glucose, 1 g/dl soluble starch, 0.5 g/dl yeast extract, 0.5 g/dl peptone and 0.1 g/dl calcium carbonate (pH 7.0 before sterilization) is used as a first seed medium. The seed culture (the first seed culture--the fourth seed culture) is carried out in the same manner as described in the Example 1. The composition of the second seed medium--the fourth seed medium is the same as that of the first seed medium. The thus obtained fourth seed culture is subjected to fermentation in the same manner as described in the Example 1. The fermentation medium has the same composition as that of the fermentation medium described in the Example 1.

After the completion of fermentation, the culture liquor is subjected to the same purification and isolation method as described in the Example 1 to obtain 130 mg of a free base of pure Fortimicin KG₁, 390 mg of a free base of pure Fortimicin KG₂ and 200 mg of a free base of pure Fortimicin KG₃.

EXAMPLE 4

In this example, the same procedure as described in the Example 1 is repeated except that Micromonospora olivoasterospora CS-26 (NRRL 8178) (FERM-P No. 3567), which is a mutant strain derived from Micromonospora olivoasterospora MK-70 (ATCC 21819) (FERM-P No. 1560) by means of treatment with nitrosoguanidine, ultraviolet irradiation and γ-ray irradiation, is used as the seed strain. As the result, 170 mg of a free base of pure Fortimicin KG₁ 450 mg of a free based of pure Fortimicin KG₂ and 280 mg of a free base of pure Fortimicin KG₃ are obtained.

EXAMPLE 5

In this example, each 1 g of the free base of Fortimicin factors KG₁, KG₂ and KG₃ is dissolved in a small amount of water. The solution is adjusted to pH 4.5 with 6 N-sulfuric acid. To the solution is added about 10 times the volume of the solution of acetone to precipitate respectively sulfates of Fortimicin factors KG₁, KG₂ and KG₃. The precipitate is taken by centrifugation and dried to obtain about 1.6 g of sulfate of Fortimicin KG₁ (620 u/mg), about 1.6 g of sulfate of Fortimicin KG₂ (620 u/mg) and about 1.5 g of sulfate of Fortimicin KG₃ (650 u/mg) respectively as a white powder. 

What is claimed is:
 1. A process for producing at least one of three Fortimicin factors of the general formula: ##STR8## wherein R is ##STR9## or H, when R is H, one of said factors having the following physicochemical properties:(1) Melting point: 95°-98° C.; (2) Specific rotation: [α]_(D) ²² =+58.3° (c=0.66, H₂ O); (3) Infrared absorption spectrum (KBr): 3360, 2930, 1675, 1590, 1370, 1110, 1055 and 1000 cm⁻¹ ; and (4) The CMR spectrum [deuterium oxide solution (pD=10.7)]: δ (ppm): 153.1, 101.2, 95.2, 83.4, 83.2, 75.2, 73.4, 62.2, 60.1, 54.8, 48.9, 47.4, 33.6, 25.7, 20.5;and another of said factors having the following physicochemical properties: (1) Melting point: 83°-85° C.; (2) Specific rotation: [α]_(D) ²² =+30° (c=0.76, H₂ O); (3) Infrared absorption spectrum (KBr): 3350, 2920, 1675, 1590, 1370, 1090, 1030 and 990 cm⁻¹ ; and (4) The CMR spectrum [deuterium oxide solution (pD=10.6)]:δ (ppm): 153.1, 101.3, 95.3, 82.2, 80.0, 71.3, 71.1, 61.1, 59.3, 53.8, 48.9, 47.3, 35.4, 25.6, 20.5;and when R is ##STR10## the remaining factor having the following physiocochemical properties: (1) Melting point: 135°-138° C.; (2) Specific rotation:[α]_(D) ²² =+185° (c=0.265, H₂ O); (3) Infrared absorption spectrum (KBr): 3370, 2940, 1640, 1580, 1462, 1110 cm⁻¹ ; and (4) The CMR spectrum [deuterium oxide solution (pD=1.2)]: δ (ppm): 168.8, 146.7, 99.5, 95.9, 76.1, 73.9, 71.4, 66.5, 57.3, 54.2, 52.9, 49.6, 47.2, 41.4, 32.8, 22.2, 16.9;which comprises culturing a microorganism belonging to the species Micromonospora olivoasterospora and capable of producing at least one of said factors in a nutrient medium until substantial antibacterial activity is detected in the culture liquor and thereafter isolating at least one of said factors therefrom.
 2. A process according to claim 1 wherein Fortimicin KG₁ having the formula: ##STR11## and the following physicochemical properties: (1) Melting point: 95°-98° C.,(2) Specific rotation: [α]_(D) ²² =+58.3° (c=0.66, H₂ O), (3) Infrared absorption spectrum (KBr): 3360, 2930, 1675, 1590, 1370, 1110, 1055 and 1000 cm⁻¹ and (4) The CMR spectrum [deuterium oxide solution (pD=10.7)]:δ (ppm): 153.1, 101.2, 95.2, 83.4, 83.2, 75.2, 73.4, 62.2, 60.1, 54.8, 48.9, 47.4, 33.6, 25.7, 20.5is isolated from said culture liquor.
 3. A process according to claim 1 wherein Fortimicin KG₂ having the formula: ##STR12## and the following physicochemical properties: (1) Melting point: 83°-85° C.,(2) Specific rotation: [α]_(D) ²² =+30° (c=0.76, H₂ O), (3) Infrared absorption spectrum (KBr): 3350, 2920, 1675, 1590, 1370, 1090, 1030 and 990 cm⁻¹ and (4) The CMR spectrum [deuterium oxide solution (pD=10.6)]:δ (ppm): 153.1, 101.3, 95.3, 82.2, 80.0, 71.3, 71.1, 61.1, 59.3, 53.8, 48.9, 47.3, 35.4, 25.6, 20.5is isolated from said culture liquor.
 4. A process according to claim 1 wherein Fortimicin KG₃ having the formula: ##STR13## and the following physicochemical properties: (1) Melting point 135°-138° C.;(2) Specific rotation: [α]_(D) ²² =+185° (c=0.265, H₂ O); (3) Infrared absorption spectrum (KBr): 3370, 2940, 1640, 1580, 1462, 1110 cm⁻¹ ; and (4) The CMR spectrum [deuterium oxide solution (pD=1.2)]:δ (ppm): 168.8, 146.7, 99.5, 95.9, 76.1, 73.9, 71.4, 66.5, 57.3, 54.2, 52.9, 49.6, 47.2, 41.4, 32.8, 22.2, 16.9is isolated from said culture liquor.
 5. A process according to claim 1 wherein said microorganism is selected from the group consisting ofMicromonospora olivoasterospora ATCC 21819, Micromonospora olivoasterospora ATCC 31009, Micromonospora olivoasterospora ATCC 31010 and Micromonospora olivoasterospora NRRL
 8178. 